A rapid and sensitive PCR assay for detection of HIV-1 specific DNA and RNA in culture supernatants and viral RNA in serum was developed. Culture supernatant from H9 and U937 cells infected with 100ng of p24 antigen /50 million cells was analyzed on a daily basis for viral p24 antigen, DNA and RNA. For analysis of DNA, culture supernatant (SUPE) was heat treated and an aliquot was used for PCR. Primer pairs from the gag, env and nef region of the viral genome were used for co-amplification. For detection of RNA, samples were treated with a guanidine thiocyanate based buffer followed by phenol/CHC13 extraction and precipitation with isopropanol. PCR-products were analyzed by slot-blots or liquid hybridization with radiolabelled oligonucleotide probes followed by PAGE and autoradiography. By this method between 1-10 copies could be detected using the 8E5 cell standard. Viral RNA present in 100 ul of serum from an AIDS patient or an equivalent of 0.2 pg of p24 antigen could be detected in culture supernatant from infected H9 cells. Viral DNA and RNA in was detected in culture supernatant of infected cells at 1 day post-infection while significant levels of p24 antigen were not detected until 2-3 days at the dose of virus used. In culture supernatants from cells treated with AZT, no viral RNA or DNA was detected at 3 and 7 days post infection and treatment suggesting that PCR on SUPES may be to monitor antiviral activity. No reactivity was observed in control samples of serum or uninfected H9 cell culture fluid. Our results suggest that PCR on culture supernatants may be useful in monitoring co-cultures from infected individuals or patients undergoing therapy, as well as to monitor infection in in vitro studies. This methodology was applied to analysis of other body fluids such as urine and sweat. The presence of virus in saliva of infected individuals is being investigated using these techniques. We are currently assessing non isotopic systems that would achieve single copy detection in conjunction with these techniques. A manuscript is in preparation and a poster presented at the VII Int. Conf. AIDS.
